AU631696B2 - Novel polypeptides, the DNA sequences allowing their expression, method of preparation, and utilization - Google Patents

Abstract

The present invention concerns a DNA sequence coding for a polypeptide with enantioselective amidase activity.

Description

5845/8 631696 S F Ref: 147817 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE: Class Int Class Complete Specification Lodged: Accepted: Published: Priority: Related Art: 44 4644 '44' 4 4' 4 4 444$ 4 44 Name and Address of Applicant: Rhone-Poulenc Sante avenue Raymond Aron 92160 Antony

FRANCE

Address for Service: Spruson Ferguson, Patent Attorneyis Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia ~4S Complete Specification for the invention entitled: Novel Polypeptides, The DNA Sequences Allowing Their Expression, Method of Preparation, and Utilization The following statement is a full description of this invention, including the best method of performing it known to me/us 0 ~8 432 141190 5845/9 L I "I NOVEL POLYPEPTIDES, THE DNA SEQUENCES ALLOWING THEIR EXPRESSION, METHOD OF PREPARATION, AND UTILIZATION

ABSTRACT

0 9 The present invention concerns a DNA sequence coding for a polypeptide 0 00 with enantioselective amidase activity.

6 0 S 04 0 0 0 1990-11-12 17:42 CREIINET REGIMEERU 33 1 45012380 P.02

IA

NOVPEL POLIPEPTIDES, THE DNA SEQUENCES ALLOWING THEIR EXPRESSION, METHOD OF PREPARATION, AND UTILIZATION The present invention concerns polypeptides that possess an enantioselective amidase activity. It also concerns the genetic material required for the expression of these polypeptides as well as a microbiological procedure for their preparation. Finally, this invention 1 0 concerns the utilization of these polypeptides and of transformed microorganisms for the enantioselective synthesis of acids from racemic amides, and in particular propionic acids, especially (S)-2-aryl-propionic acids and (R)-2-aryloxy-propioni c acids, Due to the presence of an asymmetric carbon atom, numerous 1 5 molecules possess two distinct stereolsomeric forms, R and S, one being a mirror image of the other. This is the case for the 2-aryl-propionic acids.

Most- of the time, these molecules exist as a racemic mixture, with the two isom-ers present in inore or less equal proportions. In certain cases, only one specific isomer is required, and it is therefctre practical to have a 20 means of separating the two isomers, or of performing a stereospecific synthesis of the desired isomer.

The present Invention concerns the domain of polypeptides capable of hydrolyzing amides in an enantloselective manner, in particular, racemic 2-atryl-propiontamides to (S)-2-aryl-propionic acids, and racemic 2- O' 4 2 5 aryloxy-propionarnldes to (R)-2-aryloxy-propionic acids.

*44 1 444 1Among the microorganisms in which this enzymatic activity has been demonstrated, strains of the genus Brv~atrun and 4Corvneb acteiu stand out (European patent No. 89 400197.3), and in particular, Brgvibacteriumn strain R312 (CBS 717.73). In addition, strains 3 0 such as Rhodpicocctt possess this enzymatic tcti"Vitv.

The present invention involves the characterization and purification of these enantloselective amidase activities, as well as the cloning and sequencing of the gen., ic material responsible for their expression. In that which follows, the termy "Amd" is used to designate 3 5 all enantioseleetive amidase activities. The term "Amnd sequence"' 1990-11-12 17:43 CABINET REGIMBEAU 33 1 45012880 P.03 2 designates all nucleotide sequences coding for said amidase activities.

In particulat, the objective of the present invention is to obtain high levels of expression of these enantioselective amidases in different host organisms by using recombinant DNA techniques.

One of the goals of the invention therefore concerns the DNA sequences coding for these polypeptides with enantioselective amidase activity, especially with regard to racemic 2-aryl-propionamides. In a preferred embodiment of the invention, the object concerns the nucleotide sequence coding for the enantioselective amidase of I 0 Brevibacterlum R312 (represented in Figure 8) or the enantioselective amidase of Rhodc s (represented in Figure 13), as well as any degenerated sequences coding for the same polypeptide. The invention also concerns the sequences that hybridize with these DNA sequences or with fragments thereof and which code for polypeptides with S1 5 enantioselective amidase activity. The invention also concerns the genes containing these DNA sequences.

Studies of the homology between the peptide sequences of these amidases reveal a highly conserved region responsible for the observed activity. This region corresponds to amino acids 137 to 193 of the peptide oOo 20 sequence shown in Figure 13 (nucleotides 618 to 788), and to amino acids 159 to 215 of the peptide sequence of the anidase of Brevibateium. R312 previously described, with which it shares a strict homology (67 One of the objects of the present Invention therefore concerns a DNA sequence such as that described previously, characterized by the fact 2 5 that it contains at least the sequence coding for amino acids 137 to 193 in Figure 13, or 159 to 215 in Figure 8, or a peptide sequence with at least 50 homology to these.

In particular, one of the objects of the present invention concerns a DNA sequence characterized in that it contains all or part of the Amd 3 0 sequence presented in Figures 8 and 13, or a variant thereof. For the purposes of the present invention, "variant" is meant to describe all sequences that conserve the properties of the initial sequence, even if they contain alterations resulting from, for example, mutations, deletions, insertions, or degeneracy of the genetic code.

3 5 More precisely, the DNA sequence contains the sequence presented 1_ 3 in Figures 8 or 13.

These sequences can be obtained by diverse methods. The general strategy is to clone the genomic DNA fragment coding for the desired polypeptide, with the aid of nucleotide probes derived from the purified polypeptide. By using different methods including primer elongation, restriction enzymes, insertion of adaptors, or ligation of linker oligonucleotides, a nucleotide insert containing the desired DNA sequence can be constructed. It can then be mapped and sequenced by techniques described in the literature.

1 0 Other techniques can be used as well, including the utilization of DNA and/or partial or total chemical synthesis. These techniques are well known, and the structures described in Figures 8 and 13 allow the isolation of an equivalent sequence, in any microorganism, using classical techniques.

15 In effect, having demonstrated the homology between the different 1 enantioselective amidases, the present invention allows for the Sproduction of probes that can serve to identify hybridizing genes genes with a sufficient homology) in any genomic bank. It is then easy to verify that such genes code for an enantioselective amidase. In this 20 manner, it is possible to obtain high quantities of amidase in any microorganism. It is also possible that novel enantioselective amidase activities will be revealed.

The present invention also concerns the polypeptides possessing an enantioselective amidase activity, that contain at least one of the 2 5 following peptide sequences: sequences corresponding to amino acids 137 to 193 in Figure 13 sequences corresponding to amino acids 159 to 215 in Figure 8 sequences sharing at least 50 homology with these sequences.

Another object of the invention concerns novel Dolvoentides _4purification by chromatography and gel filtration. Details are given in the examples.

More precisely, the invention concerns the enantioselective amidases of Brevibacterium R312 and Rhodococcus.

The invention also concerns transformed microorganisms containing at least one expression cassette for the DNA sequences mentioned above. These cassettes will preferably be comprised of a DNA sequence according to the present invention, placed under the control of regulatory DNA sequences that insure its expression in the desired host.

1 0 The cassette can be integrated in the host genome, or inserted in a plasmid carrying a selectable marker and an origin of replication functional in the host.

One of the interests of the present invention is the expression of these polypeptides under artificial conditions, i.e. the expression of a 6400 15 heterologous sequence in a certain cell whose culture conditions are t *"AI particularly advantageous, and/or the expression of a homologous o4, sequence under the control of at least partially heterologous regulatory signals in order to increase the production and/or ameliorate the culture conditions.

20 The DNA sequences controlling the expression of the DNA sequences that are the object of the present invention preferable carry a transcription and translation initiation region. This region contains a promoter and a ribosome binding site that can be homologous or i 2. heterologous to that of the peptide product.

l 25 The choice of regulatory region depends on the host to be used. In particular, for prokaryotic hosts, the heterologous promoter can be chosen from among the strong bacterial promoters, such as the promoters of the

S

r tryptophan operon Ptrp, the lactose operon Plac, the right or left Cot promoters of bacteriophage lambda PR and PL, the strong promoters of 1990-11-12 17:43 CRBINET REGIMBERU 33 1 45012880 P.04 enolase (ENO).

When the host microorganism is prokaryotic, the sites of ribosome fixation will preferentially be derived from either the cll gene of lambda or from homologous genes of corynebacteria.

A transcription and translation termination region functional in the host will be placed 3' to the coding sequence. The plasmid will also carry one or several markers permitting a selection of the recombinant host. Dominant markers are preferred, such as those conferring resistance to antibiotics like ampicillin or streptomycin, or to other toxins, The host microorganisms to be used notably include enterobacteria such as coli and corynebacteria of the genus Crt .nebaterium, Brevibacterium, or Rhodococcus.

Of course, other cell types can be used, based on the same principle.

One object of the invention concerns the plasmids previously 15 described containing at the least a transcription and translation initiation region, a DNA sequence coding for the desired polypeptide, and a selectable marker.

The invention also concerns the transformed microorganisms 52o previously described, regarding their application in the preparation of 20 enantioselective amidases as well as their use for enantioselective synthesis of acids from racemic amides.

The procedure for preparation of enantioselective amidases involves cultivation of the previously described microorganisms under conditions allowing expression of the sequence coding for the enantioselective amidase, followed by separation of the microorganisms from the amidase that-has been produced.

More precisely, the invention concerns the utilization of the recombinant microorganisms or polypeptides already described, for the enantioselective synthesis of 2-aryl-propionic acids from the 3 0 corresponding racemic 2-aryl-propionamides.

According to one of the preferred embodiments of the present invention, a recommendeo procedure is described that consists of the preparation of a stereoisomer of an organic acid from the corresponding racemic amide, characterized in that the racemic amide is placed in the 3 5 presence of the microorganism transformed as previously described, or in i 6 the presence of a polypeptide obtained as previously described, and the resulting stereoisomer is recovered.

Among the amides that can be subjected to this process, the racemic amide of ketoprofen should be mentioned, from which ketoprofen useful in the pharmaceutical industry can be prepared.

The examples and figures that follow present other characteristics and advantages of the present invention. These should be considered as illustrative and non-limiting.

1 0 DESCRIPTION OF FIGURES Figure 1: A. Peptide sequences (N-terminal and internal) obtained from the purified amidase from Brevibacterium R312.

1 5 B. Oligonucleotide probe derived from the internal peptide fragment.

Figure 2: h A. Strategy for the design of probe Sq 918, from the N-terminal 2 0 peptide fragment derived from the amidase of Brevibacterium R312.

B. Specific probe Sq 918.

Figure 3: A. Hybridization profile of probe Sq 918 with total genomic DNA from Brevibacterium R312 digested with EcoRI, HindIII, KpnI PstI SmaI and Sph.

B. Hybridization profile of probe Sq 762 with total genomic DNA from Brevibacterium R312 digested with BamHI, BglII EcoRI, KnI 3 0 Pst, Sall, SmaI, SphI SstI and XhoI.

Figure 4: Restriction maps of plasmids pXL1650 and pXL1651.

7 Figure Restriction map of the 5.4 kb PstI fragment containing the enantioselective amidase gene of Brevibacterium R312.

Figure 6: Sequencing strategy of the BamHI PstI fragment containing the enantioselective amidase gene of Brevibacterium R312.

Figure 7: 1 0 Analysis of the open reading frames of the sequenced fragment.

Figure 8: Nucleotide and peptide sequences of the enantioselective amidase gene of Brevibacterium R312.

Figure 9: 0 4 00 Figure o O 20 Restriction map of plasmid pXL1751.

Figure 11: 2Restriction map of plasmid pXL1752.

Figure 12: 12.5 SDS-polyacrylamide gel after Coomassie blue staining, showing the expression of the enantioselective amidase of Brevibacterium R312 in strains E. coli B and E. coli K12 E103S. Each lane corresponds to a quantity of protein equivalent to 60 jtl of the 1990-11-12 17:44 C[BINET REGIMBEAU 33 1 45012880 8 Nucleotide and peptide sequences of the enantioselective amidase gene of Rhodococcus (ParHi fragment from plasmid pXL1836).

igutoij: Restriction map of shuttle vector pSV73.

Restriction map of expression plasmid pYG811B.

Eigiuma Restriction map of expression plasrid pYG817B, 1 5 Restriction map of expression plasmid pYG822.

Plasmid pXL1029 has been described in Jung et al. (1988), A.nn Inst. Pasteur/Microbiol, 139,129-146). It carries an F-Nd-I fragment containing PRcIts-RBSdIIAtRI Idein~lfi.eaW~on atld prification of he Sonantio. ekctjgjEmid~s.f 4 t ti U1 Xdificalin (R,)-2-(4-hydroxy-phenoxy)-propionam-de (H-PPAmide), a Q: G' derivative of 2-aryloxy-propionamide, is a better substrate for the enantioselective amidase than 1-1 iE01-2 17:44 CABINET PEIBEU .33 1 4=5012680 P.06 9 reaction was carried out at 25'C with agitation in a buffer of 50 m-M sodium phosphate, pH- 7.0, in th~e presence of IDr~vibacterhliLm R312; it was stopped by addition of a mixture of 0.05 M phosphoric acid, acetonitrile, and 1. N 1-ICI in a ratio of 55/40/5 After centrifugation of the culture the supernatant was analyzed by reverse phase high performance liquid chromatography (HPLC) (FE'bar-Merck RP-18, 5 gim). Elution was performed with a solution of 0.005 M phxosphoric acid and acetonitrile (85/15) The respective concentrafidns of HPPAmide and HPPAcid were measured by comparing the elution pea-ks to a standard. For this 1 0 substrate, the enantiou ric excess is defined as (R S) x 100 where R and 8 are the respective concentrations of the R and S enantiomers of HPPAcid. The enantiorneric excess was deduced either from polariinetric measurement (using the absorption of sodium at 589 nm), or by HPLC using a chiral column.

2 1 5 The activities obtained with whole cells and a soluble extract, respectively, were 15 U/mg and 24 U/mg of protein, (1 U 1 A.mol HPPACId formed per hour) The enantiomeric excess of (R)-HPPAcid is %.These results demonstrate that Br?,yi~agjg~tfrn R312 possesses an enantioselective amidase capable of hydrolyzing racemic 2-arylpropionamides to the corresponding S acids. This was verified by the hydrolyses of racernic 2-phenyl-propionanmide and raceinic 2-(3-benzoylphenyl)-propionamlde to the reipecr~ve corresponding S acids, with an enantion-teric excess higher than 93% 1. uifiaio The purification was carried out at VCC. Cells (40 g dry weight Brevibactarjum R312) were thawed and suqpended in 300 ml Buffer A 0 Z mM sodiumn phosphate, pH 7, 5 mM j3-mertaptoethanol). Cells were then 00 broken by sonication and membrane debris were eliminated by 03 0 centrifugation at 20000 rpm for 30 minutes. To 30 xial of supernatant, ml of a 10 solution of streptomycint sulfate was added slowly, with stirring. After 45 minutes, the solution was clarified as above and the resulting supernatant was treated with amimonium sulfate. The protein fraction precipitating between 30.8 and 56.6 saturation of ammoniumn 3 5 sulfate was collected by centrifugatioti and dissolved in 35 ml 1990-11-12 17:45 CRBINET REIMBERII J140180 P0 33 1 45012880 P-07

I'

Buffer A, and then dialyzed slowly against the same buffer, The solution thus obtained was adjusted to 20 satUration of amnmonium sulfate, centrifuged, then applied to a phenyl.Sepharose CL-413 column (Pharmacia) equilibrated with Buffer A at 20 saturation of ammoniumn sulfate, Active fractions were eluted with th same buffer, then concentrated by ultrafiltration to a volume of IS ml using an Amicon lDiaflo PM10 cell. Glycerol (10 was t]~en added to the concentrated solution, and the resulting solution was applied to an U.ltrogel AcA 44 column (IBF-Biotechrdcs, France) previoutsly eq~uilibratedl with 50 m1\41 Tris-Il, pH 7, 100 mnM NaCl, Fractions containing the highest specific activity (approximately 32 of the total activity loaded onto the column) were collected, concentrated, and subjecteO3 to a supplementary filtration step on the same gel. In parallel, fractions cont~dning the highest specific activity (approximately 30 of the total protein loaded onto the column) 1 5 were analyzed by SDS-PAGE and stored. The enantioselectivity of the 44 purified protein was also determined.

This purification method resulted in an enzyme more than 80 1 7 o pure, with a specific activity of 815 U/mg. At this step, a major band of apparent molecular weight 59 5 IKt which corresponds to at least 80 4 20 of the total proteins, is visible on SIDS-PAGE. Moreover, the arnidase activity eluted from. an I-PLC TSK 3000 column corresponds to a molecular weight of 122 KD, Indicating that the enzyme is in a dimeric f orm, Table I shows the characteris tics ot the different fractions. This 25 table describes the different steps of the purification of the onantloselective anrddase of p i~a Lter~u-n4 R312: -from 40 g of humid cells, after precipitation with streptomnycin sulfate -one unit corresponds to I pgmol HPlPAcid formed per hour under the conditions described below.

~ipT~T~ I)~L-U^~IIIIIWIII*Uli i~ Table 1 SPurification Vol. :Quantity of :Activity Yield Purification Step (ml) :protein (U/mg) Factor 1/ Crude extract 325 1918 26.4 100 1 2/ Ammonium sulfate 29.5 613 62.5 :75 2.4 precipitate Phenyl-sepharose :77 200 198 :78 eluate 4/AcA44, :6 27 457 :24.4 17.3 first eluate 5/AcA44, 3 3.9 815 6.3: 31 second eluate Cot o ;a 0 a to 0 t I 60 #O I EXAMPLE 2 Clonine the enantioselective a or 90 0 0~r 0 9$ 00~ 010 a;P amidase of Brevibacterium R312 i~- 2.1 Derivation of protein sequences The peptide sequences corresponding respectively to the Nterminal extremity (27 residues) and a trypsic internal fragment (21 residues) of the enantioselective amidase of Brevibacterium R312 were determined using the purified enzyme.

This was done by subiectine 3 nmnol of thp amidaca nrrrarnrin fA' 12 this manner. To obtain the additional internal sequence, the same quantity of protein was digested with trypsin. The reduced and carboxymethylated fragments were then separated by reverse phase HPLC (2.1 x 10 mm, flow 0.2 ml/min) using the following elution buffer: a gradient of 0 to 50 acetonitrile in 0.07 trifluoroacetic acid. The peptide eluting in a well-separated peak (at 40.8 acetonitrile) was sequenced (Figure 1A).

2.2 Construction of the nucleotide probes 1 0 Two strategies were pursued.

In the first strategy, a 29-mer probe (59 minimal homology) was constructed, keeping in imind the codon usage in the tryptophan operon of Brevibacterium lactofermentum (7.7 kb sequence containing 6 cistrons: 4O. Matsui et. al., Mol. Gen. Genet. 209 p. 299, 1987), and using th& sequence 1 5 IDGALGSYDV of the internal fragment (presenting a smaller average degeneracy). The noncoding strand was synthesized with consideration of the relative thermodynamic neutrality of G:T pairing and by introducing 'ooo several degeneracies in order to maximize the average theoretical frequency of codons considered (88 in relation to the usage of the chosen codons). These considerations led to a GC content in the probe of about 69 The sequence of the probe (Sq 762) is shown in Figure 1B.

In the second strategy, the PCR method described by Girges et. al.

(Nucleic Acids Res. 16, p. 10371, 1988) was used to obtain an exact o 4; nucleotide probe from a peptide corresponding to highly degenerated codons. To accomplish this, 25-mer oligonucleotides (see underlined CO.. sequences in Figure 2A) were synthesized, corresponding to all the possibilities of coding of the first or last five codons of the N-terminal peptide sequence, and carrying EcoRI and HindIII sites respectively, at their 5' extremities. These 25-mers were used to prime an enzymatic 30 amplification of Brevibacterium R312 genomic DNA. After 30 cycles of amplification the candidate fragment was purified on a gel, then inserted between the HindIII and EcoRI sites of bacteriophage M13mp19. In fact, two different hybridization temperatures of the primer (45 0 C and 48 0

C)

were used, resulting in two candidate fragments. Thus after cloning the 3 5 fragments, several clones from each temperature were sequenced and 13 compared. The results are shown in Figure 2A. It can be seen that apart from the degeneracies introduced by the primers, a DNA fragment (unique between primers) coding for the N-terminal extremity of amidase was well amplified. A 40-mer synthetic oligonucleotide (Sq 918) corresponding to this internal fragment was therefore used for the rest of the clonage as an exact probe for the N-terminal extremity of amidase.

Figure 2B shows the nucleotide sequence of specific probe Sq 918.

The two probes Sq 762 and Sq 918 thereby obtained were labeled by phosphorylation with 32 P.

2.3 Cloning of the gene encoding the enantioselective amidase of Brevibacterium R312 The strategy consisted of first verifying the specificity of the probes and determining the nature of the genomic DNA fragment to be cloned by Southern blot. Briefly, Brevibacterium R312 genomic DNA was alternatively digested by several restriction enzymes corresponding to possible cloning sites, and in particular to sites present in the multisite cloning region of pUC plasmids. Notably, PstI was used. After electrophoresis through an agarose gel and transfer to a nylon membrane, 2 0 the various digestions were hybridized to probes Sq 762 and Sq 918. The results shown in Figure 3 demonstrate that the two probes present a sufficient specificity under the conditions of hybridization (at most one fragment hybridizing for each digestion). Furthermore, since the two probes give almost the same profile of hybridization, one might be led to believe that the hybridization signals of the sought-after gene are rather specific, and that the internal peptide obtained after trypsic digestion is very close to the N-terminal extremity. In particular, the hybridization footprints reveal the existence of a unique 5.4 kb PstI fragment that hybridized strongly with the two probes. It was therefore decided to clone 3 0 this fragment.

For the cloning, all fragments of approximate size between 4.6 and kb and 5.5 to 6.5 kb resulting from the PstI digestion of total genomic Brevibacterium R312 DNA, were purified on agarose, electroeluted, and ligated to pUC19 cut with PstI. After transformation of E. coli strain DH5a, 500 white colonies were obtained on X-gal medium, which L -1 14 theoretically correspond to recombinant microorganisms. Each colony was individually isolated, transfered onto a nylon membrane, then analyzed by hybridization with the 32 P-labeled Sq 918 probe. Two clones hybridized very strongly; they were isolated and used in following steps.

The two recombinant plasmids pXL1650 and pXL1651 isolated from these two clones were analyzed by various methods, including restriction mapping, partial sequencing using the probes as sequencing primers, and Southern blot. Figure 4 shows that the two plasmids contain the same 5.4 kb PstI insert, in the two orientations. Figure 5 shows the restriction map 1 0 of this fragment. These two plasmids indeed contain the sequences coding for the characterized peptides, the tryptic fragment adjoining the N-terminal (Figure Furthermore, these results show that the gene coding for the enantioselective amidase of Brevibacterium R312 is located on a 2.3 kb BamHI-PstI fragment, oriented in the sense BamHI toward PstI. Given the position of the 5' extremity of the coding sequence and knowing that the enzyme is coded by at most 2 kb (57 63 KD monomer according to our estimations), it is certain that the entire gene was contained in the BamHI-PstI fragment that we therefore proceeded to sequence.

EXAMPLE 3 Sequence of the BamHI-PstI fragment containing the gene encoding the ooo* oenantioselective amidase of Brevibacterium R312 0444 The sequencing strategy for the BamHI-PstI fragment is shown in Figure 6. The various sequences were all obtained by the chain termination method (Sequenase kit in the presence of 7-deaza-dGTP; 3 5 S)-dATP) either on single stranded M13 matrices carrying subfragments, or directly on plasmid pXL1650. To this end, several t 30 specific primers were also synthesized. The average GC content of the sequence obtained is 61.5 Figure 7 presents an analysis of the open reading frames; it is seen that the open reading frame corresponding to the amidase codes for 521 amino acids, a protein of calculated molecular weight of 54671. The GC content of this open reading frame is respectively 65.8 52.5 and 70 for the I i j

-I

*000 00 0 0~ 0 b 0 0 6 0 *00 0, 00 00 0 S 0 006 600 6l 4 0 r i 0.0i first, second and third codon positions, which is a typical distribution in coding sequences of GC-rich microorganisms. Figure 8 shows the complete sequence of the BamHI-PstI fragment.

EXAMPLE 4 Expression in E. coli of the gene encoding the enantioselective amidase of Brevibacterium R312 4.1 Construction of plasmids Several plasmids were constructed in which the structural gene of amidase, containing a homologous ribosome binding site (RBS) or the RBS from the clI gene of lambda, was placed under the control of its own promoter, the promoter of the tryptophan operon, or the right temperature sensitive promoter of lambda. Plasmid pXL1650 (Figure 4) 1 5 was obtained by insertion of the 5.4 kb fragment resulting from the PstI digestion of total Brevibacterium R312 genomic DNA, into the unique PstI site of plasmid pUC19. This plasmid therefore carries the promoter of the lactose operon Plac, followed by a ribosome binding site and the structural gene encoding the enantioselective amidase of Brevibacterium R312, as well as a marker encoding ampicillin resistance.

Plasmid pXL1724 (Figure 9) contains the 2.3 kb BamHI-PstI fragment excised from the 5.4 kb PstI fragment under control of the promoter of the tryptophan operon of E. coli. In this construct, the amidase gene of Brevibacterium R312 is therefore preceded by 58 base pairs upstream of 25 the ATG codon containing its own ribosome binding site (Figure 8).

Two other constructions were made in which the structural gene encoding the enantioselective amidase of Brevibacterium R312 was placed under the control of heterologous promoters, with heterologous ribosome binding sites. These plasmids (pXL1751 and pXL1752) were obtained as follows: Plasmid pXL1724 was mutagenized by PCR in order to substitute an NdeI site (CATATG) for the ATG codon situated upstream of the amidase structural gene. Amplification was carried out using a primer corresponding to the NdeI site hybridizing with the initiation ATG codon, 3 5 and a primer corresponding to an XhoI site situated downstream of the i 190-1 1? 1745 CABHINET REGIMBEAIi 33 1 45012880 P.08 16 ATO codon. The amtplified fragment was then excised by digestion with N4ld and LhpQX.

-Utilization of promoter Ptrp.- Into plasmid pXL172'4 digested by RcR and X1l9.1, was inserted an EcoRI- NAel fragment carrying the Ptrp promoter and the ribosome binding site of the lambda cli gene in which the termination sequence tRI has been deleted, and the 5' region of the amidase structural gene (fragment Ndjei- 22hQI), This generated plasrnd pXL1751 (Figure -Utilization of promoter PRcits, 1 0 The samne strategy was employed, this time by using the -E I-Ndej I fragment from plasmnid pXL1029 containing the Ppcfts promoter and the ribosome binding site of the lambda 61i gene deleted of the termination sequence tRI. This getierated plasmici pX1.1752 (Figure 11), 4.2 Exp~ression of the amidase gene of Brevibacteriumr R312 in E. coli B and E. coi K2 E coiPlasmids pXLl751 and pXL1752 were vised to transform strains E.

coi1,and F. coli K12'EI03S, respectively, by the calcium chloride method.

Selection of recombinant microorganisms, was carried out in ampicillin medium, The expression of the enantios elective amidase of Breibctrim IR312 was measured after sonication of the cells, by SDS-PAGE of the crude fractions or, after centrifugatiori, of the pellet and supernatant. The results in Figure 12 show a high level of amidase expression, representing 2 5 up to 20%of total protein.

UilizaimoDLle enaintig electiveiiml d se -of. 13ryi bad erium R3nLf.ax the enantl eedive 5.ntheIs of 2-aryl-ppjn.ad The following strains were used in that which follows:- .coi(pXL1751) the amidase coding sequence is placed under the control of the promoter of the tryptophan operon, gli (pXL1752) amnidase is produced by raising the temperature from 3 5 30 0 C to 42"C at the end of the exponential phase (MR promoter of lambda 1990-11-12 17:46 CRBINET REGIMBERI .33 1 45012880 P-09 4 17 under control of the temperature Sensitive repressor clts).

Two control strains were also used: EQjj (pXL9O6) equivalent to E. colH (pXL1751) with the arnidase gene replaced by the gene ILI 3 E. Qjj (pXL1029) equivalent to I.cu(pXL1752) with the arnidase gene replaced by the gene ILlP.

The following procedure was used to test the activity of these microorganisms: A cell suspension grown under appropriate inducing conditions 1 0 was added to a solution containing: -hydroxy-4-phenoxy-2-propionamide (I-PZ'Am), or -phenyl-2-proplonamide (PPAm), or -the amiide of ketoprofen (KAm), for exan~ple.

The reaction mixture was then diluted in a buffer containing S 1 5 acetonitrile :N hydrochloric acid (90:10) (v/v) 1 and the cells were elimnin.ated by centrifugation. The reaction mixture was resolved by HFLC and the amidase activity was calculated, The results shown in Table 2 demonstrate the efficiency of this system.

Table 2 shows the specific activity of the anidase of 13revibactrium tc 20 R1,aspouedi .coli in inducing conditions, toward the racernic substrates JiPPAm, PPArn and KAm, as well as the enantiomeric excess of the chiral acids produced. In this expefitent, JF. Iot strains harboring plasrnids pXIJ751 (arnidase) or pXL906 (control) were grown at 37'C.

2 Table 2 E. coi strains in Specific activity Enantiomeric excess :1inducing conditions v2~30 -HPAm:* PPAM IAm IMJILEM 5 geo pXL 1751 :1300 0:4:93 96:95: pXL 1732 1300505 94:97 pXL 906 0 nd :nd nd :nd n nd 12XLI :014 0: nd :ndnd Table 3 shows the specific activity of the amidase of Brvbctru' 1990-11-12 17:47 CRBINET REGI1BEAUL 33 1 45012880 R312 (expression plasmid pXCL1 751), as produced in E.-oli grown at 28 0

C

in induced or repressed conditions, toward the racemic substrates KAm, as well as the enantiorneric excess of the chiral acid produced, Table 3 1

C

4 C t 4C'. L CL~ C C

C.

V

I

Bacterial strain :Plasidd Repressor Specific activity ee -M (1)l n 9o E. Ecoll pXL1751 -55 96 I"Trp 13 rid nd not determined. eeenantiomeric excess W,) Note -Trp ,L-tryptophane.

1 5Therefore, i,.sI strains harboring the amidase gene of 1Brevibacterium R312 (genotype Amd t can efficiently hydrclyze the following three amnides (phenotype AMD+),- -24-hyd roxy-phen oxy)-p rop ion amide (HPAm) 2-phenylbpropionamide (PPArn) 20 amide of ketoprofen (KArn).

The enantiomeric excess obtained was always greater than 93 EXALIPLE 6 Pmif icatiano1 enantioselectv arnidase of R ip-iQiua 1L Assay of enzyma ic activitv The active fraction was incubated at 30 0 C for 30 mninutes in 500 l of bu~ffer (0.1 M Tris HCI pH 7.5, 5 mnM DTT, 18 mM 2-phenylpropionan-ide), After incubation, 2 ml of a mixture of acetonitrile/HCI 3 0 IN (90/10) and then 2 ml of a mixture of 50 mM H3P0 4 /CH3CN (75/25) were added to the reaction mixture. After centrifugation at 5000 rpm for minutes, an aliquot of the supernatant was subjected to HPLC to measure the reaction products.

Column: Nucleosil 5-C18 25 cmr 3 5 Eluant; 50 mM H3P04/CH3CN (75/25) 1990-11-12 17:47 CRBINET REGINBERU 33 1 45012880 P.11 19 Loading: 10 1A Flow rate: 1 xnl/rnirt A unit of activity is defined as the ql~antity of enzyme necessary for the hydrolysis of 1 pgnfol 2-phenyl-propionamide per hour.

IL Purification proloqol 6,1 Preparation of the enzyme extrag~t 7 g of cells were suspended in 15 m 0.1 M Tris FICi pH- 7.5, 5 mM DTT, and sonicated for 15 minutes at 4'C. The crude enzyme extract was 1 0 collected by centrifugation at 50000 rpm for 1 hour, 6,2, First ion-exchange chromat±ogrgjh To 20 ml of crude extract, 20 ml of BtLffer A (25 mM Tris HIi pH- mnM DTT) was added. The sample wap injected onto a Mono Q FIR 1 5 10/10 column, (Pharmacia) equilibrated in Buffer A, at a flow rate of 3 mi/mmn. After washing the columnn, the protein-s were eluted with a 0 4 fflinear I hour gradient of 0.1 to, I M XI(C at a flow rate of 3 mI/min.

Fraction size was 6 ml. The amidase eluted in 18 mld at approximately 0.3 6.3. Secon ion-exchaxge chgrma tog-raphv The active fractions were combined 4nd concentrated to 2 nil using a Centriprep ultrafiltration system (Amicon). After dilution with 6 ml Btiffer A, 4 rnl of the sample was injected at 1 mnl /min onto a Mono Q HR 5/5 column equilibrated in Buffer A. Proteins were eluted with a linear gradient of 0 to 0.5 M KCl In Buffer A. Active fractions were combined and adjusted to 15 glycerol then concentrated to 1 ml as above.

6.4 Hydophob crmatographv S3 0 1 iml of Buffer B (0.1 MA Tris 1-id pH 7.5, 0.5 mM DTT, 1.7 M

(NH

4 2 S0 4 was added to the sample which was then injected (in two injections) onto a Phenyl-Superose HR 5/5 column (Pharmacia) at a flow rate of 0.25 mlimm. Proteins were eluted at 0.5 mi/mmn with a decreasing linear gradient of VH0142S0 4 (1.7 M to 0 MWin 25 ml. Fraction size was 3 5 ml. Active fractions were adjusted to 15 %7 glycerol then diluted to 1 ml 1_1~ ~liP~ with Buffer A.

Hydroxyapatite chromatography The sample was injected at 0.5 ml/min onto a Bio-Gel HPHT column (Bio-Rad) equilibrated with Buffer C (85 mM Tris HC1 pH 7.5, mM DTT, 10 gM CaCl 2 15 glycerol). The amidase was eluted at a flow rate of 0.5 ml/min with a linear gradient of 0 to 100 of buffer 0.35 M potassium phosphate pH 7.5, 0.5 mM DTT, 10 M CaCl2, 15 glycerol in Buffer C, in 20 minutes.

1 0 These steps allow the purification to homogeneity of an enzyme with a specific activity of 988 U/mg of protein.

The enzyme thereby obtained is present in the form of a dimer of identical subunits of apparent molecular weight 53 2 KD.

1 5 EXAMPLE 7 Cloning of the gene encoding this amidase 0 After a supplementary purification step on TSK-G3000 SW, the enzyme was subjected to sequencing. The N-terminal extremity was 2 0 inaccessible to Edman-type chemistry, and so a total trypsin hydrolysis was carried out and three HPLC fractions of the hydrolysate 123, 124 and 162 provided peptides that allowed an unambiguous sequence to be obtained.

4° From the sequence obtained from fraction 123, a 32-mer nucleotide probe was synthesized, corresponding to a mixture of 8 oligonucleotides and 2 5 containing 7 inosines in positions degenerated at least three times: Probe A (from peptide 123)

ATVDVPVPDYA

3'

GCIACIGTIGATGTICCIGTICCIGATTATGC

C C C The efficiency of this probe, labeled at the 5' end with 32 p, was tested by Southern transfer onto genomic DNA from Rhodococcus previously digested by one of the following restriction enzymes: Sst, SphI SmaL 3 5 PstI, KpnL, EcoRI, SalI and BamHI. Experimental conditions were as I i c 21 follows: hybridization buffer, 5x SSC, 5x Denhardt, 0.1 SDS, 50 mM NaPO 4 pH 6.5, 250 gg/ml salmon sperm DNA; hybridization temperatures were 50 0 C or 55 0 C (two experiments); wash conditions were 1 hour in 6x SSC at room temperature and 5 min. in 2x SSC, 0.1 SDS at 50 0

C.

Under these conditions, probe A gave strong, unambiguous signals; in particular, with the BamHI, pnl, SphI SstI, Sma Sall and PstI digestions, a single genomic band was found, strongly hybridizing to probe A. For PstI digestion, the size of the hybridizing signal to probe A 1 0 corresponds to a genomic fragment of approximately 3.2 kb.

The 3 to 4 kb PstI digestion fragments of genomic DNA were thus purified by preparative electrophoresis through agarose followed by electroelution, then ligated to plasmid pUC19 that had been cut by PstI.

After transformation of E. coli strain DH5c, 600 clones that were white on LB Amp--X-gal were repicked individually and probed with probe A by colony hybridization, in stringency conditions similar to the Southern.

The 9 clones with particularly strong hybridization signals were then analyzed by restriction of plasmid DNA. Among 6 of these clones having clearly inserted the same 3.2 kb fragment in the two orientations, 2 clones representing each orientation (pXL1835 and pXL1836) were analyzed in more detail (detailed mapping, Southern analysis), thereby confirming that the desired fragment had been obtained.

EXAMPLE 8 Sequence of the 3,2 kb Pstl fragment The complete nucleotide sequence of the 3.2 kb PstI fragment was determined for the two strands. The GC content of this fragment was 62.4 similar to the GC content of R312 (approximately 62 Analysis of 3 0 the sequence revealed an open reading frame of 1386 nucleotides (position 210 to 1595) coding for a polypeptide of 462 amino acids (calculated molecular weight of 48554) that contained the three peptide previously obtained by sequencing the trypsic fragments. This open reading frame is included in a BamHI subcloned fragment whose 3 5 nucleotide sequence is shown in Figure 13.

~t1J t f| j i i of 2-aryl-propionamide derivatives, As a consequence, the enantloselective enzymatic activity was 3 5 detected using 2-(4-hydroxy-phenoxy)-propionamide as substrate. The 1

.I

22 The 3 underlined peptide sequences correspond to the peptide fragments determined directly on the trypsic fragments of the purified enzyme (peptide 123, 124 and 162). The underlined nucleotide sequence corresponds to the (degenerated) probe used to clone the gene. The peptide sequence in italics corresponds to residues 137 to 193 that are highly conserved between the enantioselective amidases of Brevibacterium strain R312 and the strain of the genus Rhodococcus (see below).

This open reading frame represents the structural gene of the 1 0 enantioselective amidase.

EXAMPLE 9 Homologies between different amidases: identification of a sequence characteristic of amidase activity S0 aA comparison of the peptide sequences of the enantioselective amidase of R312 (Figure 8) and the amidase shown in Figure 13 shows a oOoO strong homology in about two-thirds of the sequence, between residues 150 and 300 of R312 (50 strict identity), with the homology reaching 67 between residues 159 and 215.

A search of the GENPRO gene bank for homologous sequences revealed some strong homologies between the 150 to 200 region, and the sequences of the acetamidase of Aspergillus nidulans, the indolacetamide hydrolases (IAH) of Pseudomonas syringae and Bradyrhizobium 2 5 japonicum, the tms2 protein of Agrobacterium tumefaciens, and the 6aminohexanoate-cyclic-dimerhydrolyases (ACDH) of Flavobacterium Ss strain K172 and Pseudomonas strain NK87.

Table 4 shows the homology of peptide 137-193 of the amidase described above, with the respective sites of these other enzymes resulting supernatant was treated with ammonium sulfate. The protein fraction precipitating between 30,8 and 56.6 saturation of ammonium sulfate was collected by centrifugation and dissolved in 35 ml ~urs~ 23 Table 4 Amidase homology R312 65.5 tms2 A. tumefaciens 64.3 IAH P. syringae 61.8 ACDH K172 or P. NK87) 61.4 IAH B. japonicum 54.4 Acetamidase nidulans) 47.4 This strongly conserved region is most of these enzymes (catalytic site).

likely responsible for the activity a 0000s4 00 o 0 0 4 000* 0004 O 04

P

*04 C 0004 EXAMPLE Expression of the enantioselective amidase in E. coli In order to confirm the identification of the phase coding for the 2 0 enantioselective amidase, an Ndel site (CATATG) was created by PCR at the presumed ATG codon at position 210 (Figure 13), and the fragment between this site and the Sall site at position 1683, containing uniquely the region coding for amidase, was placed under the control of signals functional in E. coli for transcription initiation (promoters Ptrp or PR) 2 5 and translation (ribosome binding site cII). The vectors thereby obtained (pXL1893, Ptrp; and pXL1894, PR-cIts) are similar to vectors pXL1752 and pXL1751 expressing the amidase of R312, as previously described.

Expression from plasmids pXL1893 and pXL1894 was studied in E. coli B and E. coli K12 E103S, respectively. A protein comigrating with the 24 1. Construction of the expression vectors These vectors are derived from replicating vectors for corynebacteria. They include a replicon of E. coli a replicon of corynebacteria a selectable marker an Amd sequence.

Vector pSV73 (Figure 14): this plasmid is derived from plasmid pSR1 of C. glutamicum (Yoshihama et. al., J. Bacteriol. 162, 591, 1985) by insertion of plasmid pUC8 containing an E. coli replicon and the kanamycin resistance gene carried on transposon Tn903.

This plasmid was used to construct the different expression vectors for the Amd sequences shown in Figure 13, notably: o 0 Vectors pYG811A and B (Figure 15). These expression vectors are S,1 5 obtained by cloning the Amd sequence contained in the Sall fragment represented in Figure 13 into the Sall site of pSV73, in both orientations.

I Vectors pYG817A and B (Figure 16). These expression vectors are obtained by cloning the Amd sequence contained in the BamHI fragment t 'f <represented in Figure 13, into the BglII site of pSV73, in both orientations.

Vector pYG822 (Figure 17). This expression vector is derived from pSV73 by inserting between the SalI and BglII sites an expression cassette containing the Amd sequence shown in Figure 13 under control of the Ptrp promoter of the tryptophan operon of E. coli.

Other cryptic corynebacterium plasmids can be used for the construction of expression vectors for the Amd sequence that are functional in corynebacteria. For example, plasmid pX18, isolated from B.

lactofermentum (Yeh et. al., Gene, 47, 301-306, 1986), ailowed the construction of shuttle vectors pYG820A and pYG820C which can replicate in Brevibacterium R312 and therefore can be used as recipients for rannin, 11 i 1990-11-12 17:48 CABINET REGIMBER~U 33 1 45012880 P.12 technique is very efficient, augmenting the frequency of transformation up to 1000-fold.

SDS-PAGE analysis of sonicated cells is used to investigate the intracellular expression of the enzxrrrte in the recombinant hosts.

This example illustrates the usage of Amd-type proteins, or the recombinant microorganisms expressing these proteins, for the enantioselective syathesis of optically active organic acids by hydrolysis of the corresponrding racernic am-ides, 1. Preparation- .&sa~ C. 0 1 1 The different strains were cultured in 2 liter erlennmeyer flasks in 4 600 ml medium, tit 2 8 1C in appropriato culture conditions with an a 4 4agitation of 150 tuxns/rnin. After termin~.tion of the culture, cells were harvested, washed in a solution of NaCI (9 4/1) and stored at -18'C.

-2 0 2. i-Phenyl-propj~IDn.I-0e as substrate The protocol is as follows: The 2-phenyl-propionamide and the cell suspension were added to a flask equipped with a stirrer, and the vollume was adjusted to 5 ml with mbA potassium phosphate buffer pH- 7.0. The flask was placed in a thermostated crystallizing dish at 251C with stirring for 1 hour. The reaction mixture was then diluted with a solution of acetonitrile/HCI (911), and bacteria and cell debris were eliminated by centrifugation, The composition in acid and amide was dettrrlned by H-PLC The results obtained in BrevIbatr.LiJim R312 and PBrevikctErIji 3 0 ilctofrwa~M (ATCC 21086) are as follows, 26 I Table

[I

ii '4

I

V III h 4 11 ii 10 Stra1A Plasmid Specific activity 14tmol/h/mg- protein Brevibacierium R312 pSV73 0.1 1 of pYG8l1A :4.3 pYG811B :5.4 B. lactofermenturn pSV73 :0 IIIpYG822 2.8 3. Racemic ketoprofen amide as substrate As shown in Table 6, it is seen that recombinant corynebacteria.

1 5 expressing the rnidase from Rhodococcus gave significantly higher activities than from control cells transformed with pSV73.

Table 6 2 0 Bacterial strain Plasmid Inducer Specific activity ee [mol/h/mg protein M Brevibact. R312 pSV73 IBN :0.01 nd i 1 pYG8311A :IBN 0.04 :96 :pYG811B IBN 0.04 :94 B. lactoferxnentum :pSV73 :IBN 0 :nd IBNAin: *pYG822 :IBN :0.02 :nd :IBNAm: r= rt-,,letermine&. fee enantiomeric excess ketoprofen).

Note IBN :isobutyronitrile; IBNA-in :isobutyramide.

Claims (2)

1. 60.*0*~ A 4 4 .4 a 1Pa r4 rao The claims defining tho Invention are as folow. 1. A DNA sequence coding for a polypeptide with enantioselective amidase activity. 2. A DNA sequence according to claim 1, characterized by the fact that it is chosen from: the sequence coding for the enantioselective amidase of Brevibacterium R312 shown in Figure 8 and the sequence coding for the enantioselective amidase of Rhodococcus shown in Figure 13 an analog of these sequences coding for the same protein but resulting from the degeneracy of the genetic code DNA hybridizing with one of these sequences or with 15 a fragment thereof and coding for a polypeptide with enantioselective amidase activity 3. A DNA sequence according to claim 1, characterized by the fact that it includes at least the sequence coding for amino acids 137 to 193 shown in Figure 13, or amino acids 159 to 215 shown in Figure 8, or a peptide sequence with at least 50 homology to these sequences. 4. DNA which contains at least the coding region of the sequence shown in Figure 8. DNA which contains at least the coding region of the sequence shown in Figure 13. 6. The gene containing the DNA sequences according to one of the claims 1 through 5. 7. Novel polypeptides resulting from the expression of a DNA sequence according to one of the claims 1 through 6, and possessing an enantioselective amidase activity. 30 8. Polypeptide according to claim 7, characterized by the sequence shown in Figures 8 or 13. 9. Polypeptide possessing an enantioselective amidase activity and presenting at least a sequence chosen among: sequences corresponding to amino acids 137 to 193 of Figure 13 '.4 5 10. P( c 11. Ti ca ul 10 se 12. M th or in 15 13. M ar ri 14. M se 20 hE 15. lvi th PI tr1 25 pr la: 16. M cb th 30 17. M bi h( 18. M ch 35 th 4 a 4 444414 a II 44 4 4ar I '41 4 "44- I1 n i I ~uW~~i~ J" i 1 7 n~r~an 28 sequences corresponding to amino acids 159 to 215 of Figure 8 sequences sharing at least 50 homology with the preceding sequences. 10. Polypeptide according to one of the claims 7 through 9, characterized by the fact that it is not of natural origin. 11. Transformed microorganism containing at least an expression cassette carrying a sequence according to one of the claims 1 through 6 under the control of DNA sequences assuring the expression of this 1 0 sequence in the host organism. 12. Microorganism according to claim 11, characterized by the fact that the DNA sequences assuring the expression of a sequence according to one of the claims 1 through 6 contain a transcription and translation initiation region. 1 5 13. Microorganism according to claim 11, in which the transcription and translation initiation region contains a promoter sequence and a ribosome binding site. 14. Microorganism according to claim 13, in which the promoter sequence and the ribosome binding site can be homologous or heterologous regarding the peptide produced. Microorganism according to claim 14, characterized by the fact that the promoter sequences can be chosen from among the strong promoters of corynebacterium phages, the Ptrp promoter of the tryptophan operon, the Plac promoter of the lac operon, the left promoter PL of phage lambda, or the right promoter PR of the phage lambda. 16. Microorganism according to claim 15, in which the promoter is chosen from among the Ptrp promoter of the tryptophan operon and the right promoter PRcIts of the phage lambda. 3 0 17. Microorganism according to claim 11, in which the ribosome binding site is derived from the cTI gene of phage lambda, or from homologous genes of corynebacteria. 18. Microorganism according to one of the claims 11 through 17, characterized in that the expression cassette is carried on a plasmid 3 5 that also carries a means of selection. I *4 j I 144111 4i it II i 29 19. Microorganism according to claim 11, in which the means of selection is a selectable marker that confers resistance to an antibiotic. Microorganism according to claim 11, characterized in that the plasmid contains the Ptrp promoter of the tryptophan operon, the ribosome binding site of the cII gene of phage lambda deleted of the transcription termination sequence tR1, the DNA coding for the enantioselective amidase gene of Brevibacterium R312, and a gene conferring ampicillin resistance. 21. Microorganism according to claim 11, characterized in that the plasmid contains the temperature sensitive right promoter of phage lambda PRcIts, the ribosome binding site of the cII gene of phage lambda deleted of the transcription termination sequence tR1, the DNA coding for the enantioselective amidase gene of Brevibacterium 1 5 R312, and a gene conferring ampicillin resistance. 22. Microorganism according to one of the claims 11 through 21, characterized in that it is chosen from among the strains E. coli Brevibacterium, Corynebacterium, Rhodococcus. 23. Microorganism according to claim 22, characterized by the fact that it is strain E. coli B or E. coli K12 E103S. 24. Procedure for the preparation of an enantioselective amidase, characterized in that the microorganism according to one of the claims 11 through 23 is cultivated under conditions that allow expression of the sequence coding for the amidase, and that after culture the microorganism is separated and the amidase is extracted. 25. Procedure according to claim 24, characterized in that the culture 26. is sonicated, fractionated with ammonium sulfate, chromatographed on phenyl-Sepharose, and subjected to a double gel filtration. 26. Procedure for the preparation of a stereoisomer of an organic acid 30 from the corresponding racemic amide, characterized in that the racemic amide is placed in the presence of a microorganism described in one of the claims 11 through 23 or in the presence of a polypeptide described in one of the claims 7 to 10, and that after the reaction the stereoisomer is recovered. 27. Procedure according to claim 26, characterized in that the amide is i
2. 1990-11-12 17:48 CABINET REGIMBEU 33 14512 P3 1 a racemic 2-aryl-propionamide and the acid is an acid. 28. Procedure according to claim 27, characterized in that the racemic 2-aryl-propionamide is the amide of ketoprofen and the acid is ketoprofen. 29. Procedure according to claim 26, characterized in that the amide is a racemic 2-aryloxy-propionamide, and the acid is the corresponding R stereoisomer. A DNA sequence coding for a polypeptide with enantloselective amidase activity substantially as hereinbefore described with reference to any one of the Examples. 31. Polypeptide possessing an enantioselective amidase activity substantially as hereinbefore described with reference to any one of I the Examples. S 32. Procedure for the preparation of an enantioselective amidase substantially as hereinbefore described with reference to any one of the Examples. 33. Procedure for the preparation of a stereoisomer of an organic acid from the corresponding racemic amide substantially as hereinbefore described with reference to any one of the Examples. DATED this THIRTEENTH day of NOVEMBER 1990 Rhone-Poulenc Sante Patent Attorneys for the Applicant SPRUSON FERGUSON e